Thermal injury represents a common form of trauma associated with significant mortality and morbidity. The end-organ effect of burn-induced systemic inflammation imposes a life-threatening problem even after successful initial resuscitation. One of the mechanisms underlying multiple organ failure is microvascular barrier dysfunction, a cellular process that has yet to be understood at the molecular level. The overall goal of this research project is to define the molecular mechanisms of microvascular leakage in thermal injury. Our initial investigation during the previous funding cycle has led to the development of unique experimental models that enable quantitative analyses of microvascular permeability. The experiments revealed a series of signaling and structural modifications in the endothelial barrier involving the contractile cytoskeleton and cell-cell adhesive interactions. As a continuing effort, we propose to extend this original investigation to a more in-depth analysis of the endothelial molecular response, with a practical view toward identifying new therapeutic targets for the effective treatment of burn edema. Our hypothesis states that fluid leak during severe burn occurs via the endothelial paracellular pathway caused by MLCK210-triggered actomyosin contraction and beta- catenin serine phosphorylation-induced VE-cadherin dissociation. Selective inhibition of the contractile elements and stabilization of junctional complexes possess therapeutic potential as alternative means to attenuate the barrier injury. This hypothesis will be tested with a multifaceted approach that integrates novel cell biology techniques into physiological experiments. Through this study we wish to achieve the following specific aims: 1) to understand the molecular mechanisms of endothelial barrier dysfunction in thermal injury, and 2) to test the therapeutic effects of endothelial cytoskeleton-junction stabilizers in treating burn edema. Information gleaned from the study will significantly advance our understanding of microvascular pathobiology following thermal injury, with the potential to be translated to clinical practice for improved patient care. PUBLIC HEALTH RELEVANCE STATEMENT Thermal injury affects more than 2 million Americans each year. Despite the remarkable improvement in critical care and wound management, systemic complications remain a major cause of mortality and morbidity in patients with severe burn. Of great concern is the development of massive edema not only locally but also in tissues remote from the wound, leading to hypovolemic shock and multiple organ failure. Intensive fluid therapy is necessary for maintaining circulatory stability during the initial resuscitation period;however, without effective approaches to stop vascular leak, excessive fluid exacerbates edema. For the past few decades, tremendous research effort has been devoted to characterizing edematogenic mediators produced during systemic inflammation. However, attempts to block these mediators as therapeutic means have met with limited success. Further studies suggest that the redundant effects of multiple inflammatory mediators and their complex interactions aggravate the poor outcome of these upstream antagonists. On the other hand, alternatively interventions that directly target the end- point cellular processes may prove to be more efficacious. The objective of this study is to define the endothelial mechanisms of microvascular leakage, an end-point cellular process that has yet to be elucidated at the molecular level under the particular condition of thermal injury. We propose to provide an in-depth analysis of the specific endothelial cytoskeletal contractile responses and cell-cell adhesive interactions that may serve as therapeutic targets in burn. Information gleaned from this research will significantly advance our understanding of microvascular pathobiology in disease and injury, with the potential to be translated to clinical practice for improved patient care.